DEVELOPMENT OF H/D EXCHANGE FOR PROTEIN BIOPHYSICS

蛋白质生物物理学 H/D 交换的发展

• 批准号: 7721414
• 负责人: MICHAEL L GROSS
• 金额: $ 1.92万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721414
• 关键词: Amides; Binding; Biophysics; Complex; Computer Retrieval of Information on Scientific Projects Database; Detection; Development; Electrospray Ionization; Funding; Gases; Grant; Institution; Ions; Label; Ligand Binding; Ligands; Literature; Mass Spectrum Analysis; Measures; Metals; Methods; Peptides; Phase; Property; Proteins; Reaction; Research; Research Personnel; Resources; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis; System; Testing; United States National Institutes of Health; stoichiometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein-ligand interactions by mass spectrometry, titrn., and H/D exchange (PLIMSTEX) is a new mass spectrometric method for detg. assocn. consts. and binding stoichiometry for interactions of proteins with various ligands, as well as for quantifying the conformational changes assocd. with ligand binding to proteins. The assocn. consts. detd. with PLIMSTEX agree with literature values within a factor of six, establishing its validity for protein interactions involving metal ions, small org. mols., peptides, and proteins. PLIMSTEX provides soln., not gas-phase, properties by taking advantage of ESI and MALDI mass spectrometry to measure accurately the mass of a protein as it undergoes amide H/D exchange. The approach sidesteps the problem of relating gas-phase abundances of the protein or protein-ligand complex ions to their soln. concns. With on-column concn. and desalting, high picomole quantities of proteins are sufficient for reproducible mass detection, and the concn. of the protein can be as low as 10-8 M. It is amenable to different protein/ligand systems in physiol. relevant media. No specially labeled protein or ligand is needed. PLIMSTEX offers minimal perturbation of the binding equil. because it uses no denaturants, no addnl. spectroscopy or reaction probes, and no phys. sepn. of ligand and protein during binding. In this research, we are developing, testing, and extending further the method.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 质谱法测定蛋白质-配体相互作用，滴定，和H/D交换（PLIMSTEX）是一种新的质谱分析方法。联合常数以及蛋白质与各种配体相互作用的结合化学计量，以及用于量化相关的构象变化。配体与蛋白质结合。 协会。常数detd.与PLIMSTEX在六个因子内与文献值一致，建立了其对涉及金属离子，小有机分子，肽和蛋白质。 PLIMSTEX提供溶液，利用ESI和MALDI质谱法，在蛋白质进行酰胺H/D交换时，准确测量蛋白质的质量。 该方法回避了将蛋白质或蛋白质-配体络合物离子的气相丰度与它们的溶液相关联的问题。conns。 柱上浓度而且，高皮摩尔量的蛋白质足以用于可重复的质量检测，并且浓度为100 μ g/ml。的蛋白质可以低至10-8 M。 它适用于生理学相关介质中的不同蛋白质/配体系统。 不需要特别标记的蛋白质或配体。 PLIMSTEX提供了最小的结合平衡扰动。因为它不使用变性剂，不使用addnl。光谱或反应探针，而没有物理分离。配体和蛋白质的结合。 在这项研究中，我们正在开发，测试和进一步扩展的方法。